Saccharides (also called sugars or carbohydrates) are main components of biological systems. Saccharides constitute about 80% of dry weight of plants and, either as monomers (monosaccharides) or polymers consisting of monosaccharides covalently bound each other (oligosaccharides), are indispensable components of metabolic pathways in higher animals. In addition, saccharides are often found as parts of larger biological macromolecules (including proteins, lipids and nucleic acids). Saccharides in such various forms have numbers of important functions in nature.
A means of identifying a free monosaccharide or a monosaccharide as a monomeric component of an oligosaccharide is very useful because of the importance of saccharides in biological systems. Furthermore, a means of identifying a monosaccharide at a reducing end of an oligosaccharide is important for the structural analysis of the oligosaccharide.
Recently, a method for analyzing a structure of an oligosaccharide was disclosed in WO 96/17824 (JP-A 11-501901). In the method, a monosaccharide at a reducing end of an oligosaccharide is converted to an N,N′-diacetylhydrazino monosaccharide derivative, and the derivative is identified by gas chromatography/mass spectrometry (GC/MS) or the like. Theoretically, the types of monosaccharides at the reducing ends of all oligosaccharides can be identified according to this method.
However, there is a problem that one can use the above-mentioned method only for an isolated saccharide. If this method is applied to a mixture of several kinds of saccharides, it is impossible to determine the saccharides from which the respective resulting several kinds of hydrazino monosaccharides derive. Thus, if a naturally occurring saccharide is to be analyzed according to this method, it is necessary to isolate the saccharide beforehand.
For example, since a protein or a nucleic acid itself has ultraviolet absorbance, it is possible to trace the position during separation/purification procedures based on the absorbance. However, since a saccharide does not have such absorbance, it is desired to label the saccharide in order to facilitate separation/purification. For example, labeling with a fluorescent dye is suitable for this purpose. If a saccharide coexists with other contaminants derived from a natural source (e.g., proteins, nucleic acids, etc.), the saccharide having a label should be readily distinguished from other contaminants. Thus, in this case, a saccharide must be selectively labeled such that contaminating components other than the saccharide are not labeled.
A method for labeling a reducing residue of a saccharide exemplifies a method for selectively labeling a saccharide without labeling other components derived from a natural source. Examples of reducing residues include a carbonyl group at a reducing end of a saccharide and a free aldehyde group. Reactions on reducing residues of saccharides include a reductive amination reaction and a hydrazidation reaction. These reactions are irreversible. A method in which 2-aminopyridine is used (S. Hase, T. Ibuki and T. Ikenaka, Journal of Biochemistry, 95, 197-203 (1984)) exemplifies a method for labeling a saccharide using a reductive amination reaction. A method in which Biotin-x-hydrazide (Calbiochem) is used (B. Ridley, D. Mohnen et al., Analytical Biochemistry, 249, 10-19 (1997)) exemplifies a method for labeling a saccharide using a hydrazidation reaction. In the latter method, a saccharide labeled with biotin through a hydrazide bond is prepared by forming a hydrazone by a reaction of a saccharide having a reducing end with Biotin-x-hydrazide and then conducting a reduction reaction.
A free reducing end of a saccharide is utilized in the method disclosed in WO 96/17824. Therefore, it is impossible to directly use a monosaccharide isolated by labeling a reducing end of a saccharide according to this method for identifying the type thereof.
Furthermore, determination of the position of binding to a neighboring monosaccharide (hereinafter also referred to as the “substitution position”) is desired in addition to identification of the type of a monosaccharide in order to precisely determine the structure of a monosaccharide at a reducing end of a saccharide. In other words, it is desired to determine the position of a hydroxyl group of a neighboring monosaccharide through which a monosaccharide at a reducing end binds. Only a technique for identifying the type of a monosaccharide at a reducing end of a saccharide is disclosed in WO 96/17824. A technique for determining the substitution position is not mentioned therein.
A methylation analysis is known as a method for determining the position of binding between monosaccharides constituting a saccharide. A general methylation analysis comprises methylation of an oligosaccharide, hydrolysis of the methylated oligosaccharide, reduction of a free methylated monosaccharide, acetylation of a methylated alditol, and an analysis of a partially methylated alditol acetate (PMAA) in this order. Fragmentation patterns for partially methylated alditol acetates upon mass spectrometric analyses and rules thereof have been studied in detail for a long time, and it is possible to identify the position of an acetyl group based on the fragmentation pattern (B. Lindberg, Methods in Enzymology, Vol. 28, pp. 178-195 (1972)). PMAAs are generated for all monosaccharides constituting an oligosaccharide according to a general methylation analysis. They are usually analyzed using gas chromatography/mass spectrometry (GC/MS) equipment. The position of an acetyl group can be identified based on the fragmentation pattern upon a mass spectrometric analysis. On the other hand, one has to rely on identification by comparison with a standard substance on gas chromatography for identification of the type of the monosaccharide. For this purpose, it is required that all possible PMAAs for all monosaccharides constituting an oligosaccharide have been provided as standard substances. It requires a lot of labor to prepare possible PMAAs for all naturally occurring monosaccharides. In addition, it is practically impossible to conduct chromatography that can be used to separate and identify all possible PMAAs for all naturally occurring monosaccharides.